Fragment-Based Screen of SARS-CoV-2 Papain-like Protease (PLpro)

Coronaviruses have been responsible for numerous viral outbreaks in the past two decades due to the high transmission rate of this family of viruses. The deadliest outbreak is the recent Covid-19 pandemic, which resulted in over 7 million deaths worldwide. SARS-CoV-2 papain-like protease (PLPro) plays a key role in both viral replication and host immune suppression and is highly conserved across the coronavirus family, making it an ideal drug target. Herein we describe a fragment-based screen against PLPro using protein-observed NMR experiments, identifying 77 hit fragments. Analyses of NMR perturbation patterns and X-ray cocrystallized structures reveal fragments bind to two distinct regions of the protein. Importantly none of the fragments identified belong to the same chemical class as the few reported inhibitors, allowing for the discovery of a novel class of PLPro inhibitors.


Experimental Procedures
All experiments conducted in this work do not have unexpected or unusually high safety hazards.
Protein Expression and Purification.
The gene containing the ubiquitin-like domain and the catalytic core of SARS-CoV-2 PL Pro (residues 1-315) was synthesized with codon optimization for Escherichia Coli and cloned in the pET28a(+) vector by GenScript.We made PL Pro constructs to optimize the protein for NMR-based fragment screen (residues 71-314 with C111S and C270S), X-ray crystallography (residues 1-314 with C111S and C270S) and the enzymatic assays (residues 1-315 with C270S) were obtained by site mutagenesis.All PL Pro plasmids were transformed into the BL21(DE3) strain E. Coli.The bacteria were cultured in Luria-Bertani broth or M9 minimal media containing 15 NH 4 Cl supplemented with 50mg/mL Kanamycin at 37 o C until the optimal density at 600nm reached 0.8 before inducing protein expression by the addition of 0.1mM IPTG and 0.1mM ZnCl 2 at 18 o C for 20 hours.The cell pellet was harvested by centrifugation at 5,000g for 15 minutes, re-suspended in lysis buffer (50mM Tris pH 7.0, 500mM NaCl, 5%glycerol, 10mM imidazole, 5mM BME, 0.1% Triton X-100 and 1mM PMSF), and lysed in APV2000 lab homogenizer (SPX flow).Cell lysate was centrifuged at 15,000g for 45 minutes and loaded onto HisTrap FF column (Cytiva).The column was washed with 10 column volumes of Buffer A (50mM Tris pH 7.0, 500mM NaCl, 5%glycerol, 10mM imidazole, 5mM BME) and eluted with Buffer B (50mM Tris pH 7.0, 500mM NaCl, 5%glycerol, 500mM imidazole, 5mM BME) using a linear gradient program from 0 to 100% Buffer B over 10 column volumes.To the fractions containing PL Pro , Thrombin was added to remove the 6xHis tag and dialyzed against Buffer A without imidazole overnight.Then, Tag-cleaved PL Pro was loaded on a HisTrap column.The flowthrough was concentrated and subjected to HiLoad 26/600 Superdex75 pg (Cytiva) and eluted using Buffer C (20mM HEPES pH 7.0, 150mM NaCl, 3mM DTT) for the NMR-based fragment screen or Buffer D (25mM Tris pH 7.0, 150mM NaCl, 3mM DTT) for X-ray crystallography and the enzymatic assays.Protein concentration was quantified by the Pierce 660nm assay (ThermoFisher).

NMR Experiments.
All NMR experiments were performed at 25 o C using a 600 MHz Bruker Avance III spectrometer equipped with a 5mm single-axis x-gradient cryoprobe and a Bruker SampleJet.Gradient-enhanced, two-dimensional 1 H-15 N heteronuclear multiple-quantum coherence (SOFAST-HMQC) spectra of PL Pro were recorded using 24 scans of 12-minute acquisition times and analyzed using Topspin 4.1.4(Bruker).Our in-house fragment library of 13,824 compounds was screened as mixtures of 12 fragments prepared in twelve 96-well plates.Each NMR sample was made of 15M of 15 N-labeled PL Pro , 800M of each fragment, and 5% DMSO-d 6 for spectrometer locking in 5mm-diameter NMR tubes.Hit mixtures were identified by comparing the chemical shifts of the backbone resonances to a ligand-free PL Pro spectrum and then deconvoluted by screening individual fragments.
SOFAST-HMQC titration experiments were used to determine binding affinity of the fragment hits identified from the screen.The changes in 1 H-15 N chemical shifts of backbone resonances upon the addition of increasing concentrations of the fragments (0.0625-2mM) were analyzed.The binding affinities (K d s) of the fragments were calculated using the Hill's equation model in Prism 10 (GraphPad).

Figure S1 .
Figure S1.Chemical shift perturbations and affinity measurement for fragments 5 and 9. (A) Sections of the 1 H-15 N SOFAST-HMQC spectrum, with the overlaid of the ligand-free PL pro spectrum (blue) and PL pro spectra with increasing fragment concentrations (0.125mM red, 0.75mM pink, 1mM green and 2mM black) (B) Titration curves of selected fragments.Reported K d s were average K d s from peaks.

Figure S2 .
Figure S2.Electron density maps of fragments 5, 7 and 11 contoured at 1 level in PL pro crystal structures.

Table S1 .
X-ray data collection and refinement statistics for fragments bind to PL pro .